224 GEORGE H. HOGEBOOM AND WALTER C. SCHNEIDER 



The mitochondria account for about 25% of the total mass or nitrogen 

 of rat or mouse liver. Their most striking biochemical property (Table II) 

 is their content of enzyme systems related to the respiratory activity of 

 the cell. Thus the terminal respiratory enzyme, cytochrome oxidase, is 

 largely recovered in this cell fraction. That cytochrome oxidase is, in fact, 

 an exclusive property of mitochondria has been indicated by the following 

 observations: when liver homogenates were fractionated in isotonic sucrose 

 solutions, 79% of the original cytochrome oxidase activity was recovered 

 in the mitochondria, 20% in the nuclear fraction, and 4% in the micro- 

 somes.^* Although the small amount of activity in the latter fraction was 

 readily explained by contamination with mitochondria, it was necessary, 

 particularly in view of Bounce's conclusion with respect to the cytochrome 

 oxidase of cell nuclei,^ -^ to obtain more complete separation of nuclei and 

 mitochondria in order to determine whether the nuclei themselves con- 

 tributed to the activity of the nuclear fraction. This was achieved by frac- 

 tionation of homogenates prepared in isotonic sucrose containing a low 

 concentration of CaCU .'* The results of several experiments are sum- 

 marized in Table V. It can be seen that approximately 90% of the nuclei 

 (as shown by DNA phosphorus determinations) were recovered in a frac- 

 tion that contained 1.1 % of the original number of mitochondria and 1.0% 

 of the original cytochrome oxidase activity. Since, in these and a number of 

 other similar experiments, the cytochrome oxidase activity of preparations 

 of isolated nuclei corresponded closely to the number of mitochondria 

 present, it was concluded that the nuclei themselves did not contain de- 

 tectable amounts of enzyme activity. The significance of the apparently 

 exclusive localization of cytochrome oxidase in mitochondria becomes 

 evident when it is realized that the ultimate source of energy for all the 

 activities of the cell is mainly provided by this enzymic reaction. 



It is not surprising, of course, that systems closely related to cytochrome 

 oxidase should also be found in the mitochondrial fraction. Thus the par- 

 ticles contain a high concentration of cytochrome c (Table IV) and are 

 capable, through the cytochrome c reductases (Table II), of transferring 

 electrons between cytochrome c and the pyridine nucleotides, DPN and 

 TPN. Neither of the cytochrome c reductases is localized exclusively in 

 mitochondria, however, but both are concentrated in the microsomal frac- 

 tion as well. That mitochondria play an important role in the Krebs cycle 

 series of reactions is indicated by the presence in the fraction of succinic 

 dehydrogenase, which appears to be an exclusive property of mitochon- 

 dria,!^ fumarase, and by the oxidation of a-ketoglutarate, oxalacetate, and 

 octanoate (Table II). There is, in addition, evidence from in vivo experi- 

 ments that the synthesis of citrate by the condensing enzyme of Stern 



